About 60% of all newborns become visibly jaundiced during the first two weeks of life. The jaundice is due to a normal, transient accumulation of the yellow pigment unconjugated bilirubin IX-α (referred to as bilirubin henceforth), a product of hemoglobin catabolism. The bilirubin accumulation is due increased bilirubin production as fetal red blood cells have shorter life spans versus adult red blood cells and delayed bilirubin excretion as the metabolic pathways for eliminating bilirubin mature over the first few days of life. Therefore, a transiently elevated blood bilirubin level, referred to as hperbilirubinemia, that is often accompanied by visible jaundice, is a normal, usually harmless event in newborns during the first few days of life. However, bilirubin is neurotoxic, and in some circumstances causes severe neurological injury resulting in death or serious sequelae, and clinicians therefore closely monitor newborns with hyperbilirubinemia.
Neurologically toxic levels of bilirubin cause a spectrum of serious neurological injuries such as acute bilirubin encephalopathy resulting in death with kernicterus at autopsy (yellow staining of specific brain nuclei) or chronic neurological sequelae (also referred to as kernicterus) including choreoathetotic cerebral palsy, high tone hearing loss, paralysis of upward gaze, and yellow staining of the teeth. In addition, there is recent concern that bilirubin neurotoxicity contributes to other neurological disorders including auditory neuropathy spectrum disorder, apnea in premature newborns, and possibly autism. This spectrum of neurological damage is collectively referred to as a bilirubin-induced neurologic dysfunction (BIND).
BIND can be prevented or ameliorated by increasing bilirubin excretion from the body using phototherapy or the more risky and invasive procedure known as blood exchange transfusion in which the newborn's blood with high bilirubin levels is slowly removed and replaced by compatible donor blood with low bilirubin levels. Clinicians currently use the serum or plasma total bilirubin concentration (BTotal) as shown in Table 1 below for newborns less than 35 weeks (see, e.g., Maisels M J. et al. An approach to the management of hyperbilirubinemia in the preterm infant less than 35 weeks of gestation. J Perinatol 2012; 32:660).
TABLE 1Gestational AgeBTotal (mg/dL)(weeks)PhototherapyExchange Transfusion<280/75-611-1428-296/76-812-1430-316/78-1013-1632-336/710-1215-1834-346/712-1417-19
The ranges of treatment BTotal used in TABLE 1 (versus using a single treatment BTotal) are based on clinical experience and expert opinion rather than evidence-based, and introduce considerable uncertainty as to when treatment is needed as illustrated in FIG. 1 where, for example, in newborns less than (<) 28 weeks gestation, exchange transfusion is considered at BTotal=11 mg/dL but not mandatory until BTotal reaches 14 mg/dL. How does a clinician decide whether a newborn less than 28 weeks gestation and a BTotal=12 mg/dL needs an exchange transfusion? The uncertainties are even greater in newborns greater than or equal to (≥) 35 weeks gestation wherein there are no mandatory BTotal for phototherapy or exchange transfusion, the latter being only being “considered” when the BTotal reaches 25 mg/dL (see American Academy of Pediatrics, Management of hyperbilirubinemia in the newborn infant 35 or more weeks of gestation. Pediatrics 2004; 114:297-316). These uncertainties lead to excessive treatment resulting in significant social and financial costs, yet this approach has not eliminated BIND.
Ranges of treatment BTotal are used, e.g. in TABLE 1 because BTotal correlates poorly with BIND (e.g. see Watchko J F et al. The enigma of low bilirubin kemicterus in premature infants: why does it still occur, and is it preventable? Semin Perinatol 2014; 38: 397-406 and Ip S et al. An evidence-based review of important issues concerning neonatal hyperbilirubinemia. Pediatrics 2004; 114: e130). Since neither phototherapy or exchange transfusion are without risk (including death), newborns may suffer from BIND or complications from unnecessary treatments.
As illustrated in FIG. 2, measuring plasma bilirubin binding is important since only the non-albumin bound or free plasma bilirubin (BFree) crosses capillaries and the blood-brain barrier to enter the tissues where the brain resides. The higher the BFree at any Btotal, the higher the corresponding tissue levels of bilirubin with greater brain exposure to bilirubin and, therefore, the risk of BIND, as illustrated in FIG. 3. Bilirubin binding is highly variable in newborn plasma, and newborns with poor bilirubin binding will have relatively higher BFree and tissue bilirubin levels at any BTotal compared to newborns with normal binding, since, when poor bilirubin is present, the accumulated bilirubin needed to reach a given BTotal is greater, and the higher tissue bilirubin levels at that BTotal, increase the brain exposure to bilirubin and the risk of BIND relative to comparable newborns with normal bilirubin binding (see FIG. 2 and FIG. 3).
Recent studies document that BIND is predicted by BFree=in newborns with hyperbilirubinemia that have similar BTotal (see FIG. 3, and e.g. Amin S B, et al. Chronic auditory toxicity in late preterm and term infants with significant hyperbilirubinemia. Pediatrics 2017; 140: e20164009), validating adding bilirubin binding to the routine evaluation of these newborns. Furthermore, bilirubin binding is routinely measured in Japan and has been reported be very helpful clinically (e.g. see Morioka I et al. Serum unbound bilirubin as a predictor for clinical kemicterus in extremely low birth weight infants at a late age in the neonatal intensive care unit. Brain Dev 2015; 37:753).
BTotal and BFree are commonly but mistakenly viewed as independent alternatives for guiding clinical care, with the misconception that BFree treatment criteria would somehow replace current BTotal treatment criteria, e.g. TABLE 1. BTotal and BFree are not independent but rather interdependent measurements, inextricably linked chemically with plasma bilirubin binding sites (e.g. albumin) through the law of mass action. The risk of BIND depends on how much bilirubin has accumulated and how it distributed between blood and tissue, which is determined by BFree (FIG. 2) which in turn is a mathematical function of the BTotal and the concentration and inherent binding ability of plasma bilirubin binding sites (e.g. albumin) as described in detail below. A workable approach for incorporating bilirubin binding into clinical care is to quantify bilirubin binding in a manner that allows identification of those newborns with below, average or poor bilirubin binding and adjusting the current BTotal treatment guidelines accordingly. This reduces the uncertainty inherent in using BTotal alone to determine the risk of BIND (e.g. FIG. 1) by individualizing care.
Quantifying plasma bilirubin binding requires determining (1) the maximum amount of bilirubin that can be bound (BTmax) and (2) how tightly it can be bound, which is typically quantified using equilibrium association or dissociation constants. BTmax depends on the concentration of functioning bilirubin binding sites and is often referred to as the bilirubin binding capacity or the BTotal at which the binding sites are “saturated” with bilirubin (e.g. if the concentration of binding sites is 453 μmol/L, BTmax=26.5 mg/dL=453 μmol/L). How tightly bilirubin is bound at a binding site is quantified by a binding constant, e.g. an equilibrium association constant Kn, where n is the number of sites with different inherent abilities to bind bilirubin, and the constants representing each site are K1, K2 . . . Kn. The chemical equilibrium is
wherein BTotal−BFree is the concentration of bilirubin bound to binding sites and BTmax−(BTotal−BFree) is the concentration of the unoccupied (available) bilirubin binding sites. Albumin is known to have at least two bilirubin binding sites, and quantifying bilirubin binding using standard methods to obtain BTmax, and the corresponding equilibrium constants requires measurement of BFree at several BTotal (see Jacobsen J. Binding of bilirubin to human serum albumin—Determination of the Dissociation Constants. FEBS Lett 1969; 5: 112-114). The significant testing time, large sample volumes, and complexity of data analysis preclude routine quantification of bilirubin binding in clinical laboratories using standard methods.